139 Does centrosome malposition and fragmentation predispose to chromosome mis-segregation during the first mitotic division of equine intracytoplasmic sperm injection zygotes?

In vitro production of equine embryos via intracytoplasmic sperm injection (ICSI) has become increasingly widespread, but the blastocyst yield in clinical practice is suboptimal (~20%). In vitro-produced embryos have been proposed to be at an elevated risk of developmental arrest due to chromosome segregation errors arising during the first postfertilization mitotic cell division. In somatic cells, the centrosomes are the microtubule organizing centres that orchestrate spindle assembly and chromosome segregation, and are composed of two centrioles surrounded by pericentriolar material. Mammalian oocytes lack centrosomes, and although two centrioles are re-introduced by the spermatozoon at fertilization they appear to play only a minor role in zygotic spindle assembly. Although not essential for spindle assembly, it is not known whether the centrosomes play a role in ensuring the fidelity of chromosome segregation during the first mitotic division. In this study, the position and integrity of centrosomes in horse ICSI zygotes was examined during the first mitotic division. Cumulus–oocyte complexes (COCs) were recovered from Warmblood mares by ovum pickup, maintained in H-SOF at room temperature overnight before maturation in vitro, and then fertilized by ICSI. After 24 h of post-ICSI culture, the presumptive zygotes were fixed and immunostained for microtubules (α-tubulin or acetylated tubulin), (peri-)centrosome material (NEDD1), and chromatin (DAPI). Confocal microscopy was performed using a Leica SPE II microscope at 63× magnification. A z-stack of images at 0.17 µm intervals was generated and analysed using the 3D image analysis software Imaris 8.0. Of 326 COCs collected, 177 reached metaphase II and were fertilized by sperm injection and cultured further. Of 136 presumptive zygotes imaged, 13 were at the 2-pronucleus stage, 11 at prophase/prometaphase, 14 at metaphase, 11 at anaphase or telophase, 54 at the 2-cell stage, and 33 were either degenerated or poorly stained. During the 2-pronucleus and prophase/prometaphase stages, the centrosomes localised at the interface between the two pronuclei. In 56% (n = 14/25) of the zygotes in pro-metaphase and metaphase, independent spindles formed around each of the two parental pronuclei. The majority of dual spindles formed synchronously and were closely adjacent. However, in 64% (n = 9/14) of metaphase zygotes, the (peri-)centrosomes were either malpositioned (i.e. not at the spindle poles) or fragmented. Fragmented or multiple centrosomes are known to predispose to abnormal chromosome-microtubule attachment and aberrant chromosome segregation. We, therefore, propose that malposition and fragmentation of the centrosomes contributes to aneuploidy and developmental arrest of equine ICSI embryos.